The rhesus blood group antigens are clinically important because of their highly immunogenic nature. Specifically they are central in the pathogenesis of Rh haemolytic disease of the new born (HDN) and some autoimmune haemolytic anemias. Furthermore in blood transfusion it is important to avoid immunisation of Rh-negative recipients, particularly women, with Rh-positive blood and to avoid transfusion of immunised patients with Rh-incompatible blood products. There are five most commonly typed Rh antigens: C/c, E/e and the D antigen which is the most immunogenic, defining an individual as Rh-positive or Rh-negative. Previously it was hypothesised that RhD may have an alternative allelic gene which was designated (d); however, Southern analysis has since shown that RhD negative phenotypes result from the absence of RhD genes that code for the D antigen as described in Colin et al Blood 78:2747 (1991). In other words RhD-positive individuals have either one or two RhD genes per cell and RhD-negative individuals have no RhD genes at all.
RhD typing was initially performed by agglutination with human polyclonal anti-D sera but has recently progressed to agglutination with IgM and/or blends or IgM and IgG anti-D monoclonal antibodies. However, even these monoclonal antibodies may not detect some weak RhD antigens and RhD variants. Additionally these serological techniques only allow a probable RhD genotype (one or two D genes) to be assigned based on Rh phenotype and available population statistical data. Often unambiguous RhD genotypic information is required such as in the case of prenatal counselling of Rh-negative mothers previously immunised with an Rh-positive child.
Reference may also be made to Lo et al. Vol 341 1147-1148 of the Lancet (1993) wherein a prenatal determination of fetal RhD status by analysis of peripheral blood of rhesus negative mothers was carried out. In this reference the authors utilised firstly the sequence of the recently cloned RhD gene (Le Van Kim et al. Proc Natl Acad. Sci USA (1992) 89 10925-29), and secondly the observation that RhD negative individuals lack this gene (Colin et al. Blood 78 2747 1991). Lo et al therefore designated a PCR assay to detect RhD DNA sequences from a RhD-positive fetus by amplification from the peripheral blood of RhD-negative mothers.
In the Lo et al. assay the controls utilised were a 1 in 10.sup.5 dilution of 1 .mu.g homozygous RhD-positive DNA in 5 .mu.g RhD-negative male DNA as a positive control and water as a negative control. The other samples assayed were clinical samples from patients as well as 5 .mu.g RhD-negative male DNA. The marker utilised was pBR322 DNA digested with HaeIII.
In the Lo et al. PCR assay PCR primers were designed to amplify regions outside one coding sequence for the D gene at the 3' end. They did this because the Rh CE gene and D genes are very closely related. They therefore chose the 3' non coding region of the D gene which differs from the CE gene. They therefore did not amplify the CE gene at all. In the PCR assay PCR products were analysed by agarose gel electrophoresis. However, this PCR assay could only indicate the presence or absence of the RhD gene in the sample tested and could not be utilised for quantifying the number of RhD genes present (ie. one or two genes). The inability to quantify the number or dosage of RhD genes present means that a true RhD genotype of an individual could not be assigned.
Reference also may be made to Arce et. al. Blood 82 651-655 (1993) which refers to molecular cloning of RhD cDNA derived from a gene present in RhD positive, but not RhD negative individuals as well as Bennett et al. The New England Journal of Medicine, Aug. 26 (1993) 607-610 which refers to prenatal determination of fetal RhD type by DNA amplification. Both these references identify the D gene but do not give a D gene dosage unlike the present invention.
In the Bennett et al. reference two pairs of primers are produced wherein a first pair of primers amplify a 136 bp region common to the RhCcEe and RhD genes (i.e. exon 7) and the second pair of primers amplify a 186 bp region specific to the 3' untranslated sequence (exon 10) of the RhD gene. The two amplification reactions are performed in the same tube. Only the 136 bp product is amplified from RhD negative DNA whereas both the 136 bp and 186 bp products are amplified from RhD positive DNA.
The Arce et al. reference amplifies a region of the D gene known as "exon 4 to 5" by experiments carried out by the inventor(s). It has also been established that the exon 10 method gives more false positives than exon 7. It has also been established that PCR of exon 4 to 5 is also subject to false positives and is hard to perform.